ADD results for il15 and integrin blocking sections

tex/thesis.tex

@@ -2279,9 +2279,20 @@ do the inverse.
 
 \subsection{blocking integrin binding does not alter expansion or phenotype}
 
+% BACKGROUND add background into why integrins are important
+
+One of the reasons the \gls{dms} platform might be performing better than the
+beads is the fact that they are composed of gelatin, which is a collagen
+derivative. The beads are simply \gls{mab} attached to a polymer resin coated
+onto an iron oxide core, and thus have no analogue for collagen. Collagen
+domains present on the \gls{dms} group could be creating pro-survival and
+pro-expansion signals to the T cells through \gls{a2b1} and \gls{a2b2}, causing
+them to grow better in the \gls{dms} system.
+
 % TODO perhaps these figs should be combined
 % TODO actually make the captions for these
 % TODO add some background into why integrins are important and the proposed mechanism
+% TODO add an experimental timeline to these showing when I added the mabs
 \begin{figure*}[ht!]
   \begingroup
 
@@ -2297,6 +2308,20 @@ do the inverse.
   \label{fig:integrin_1}
 \end{figure*}
 
+% TABLE add regression table showing the results from this analysis
+
+We tested this hypothesis by adding blocking \glspl{mab} against \gls{a2b1}
+and/or \gls{a2b2} to running T cell cultures activated using the \glspl{dms}.
+These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and
+\gls{a2b2} were known to be expressed {\#}. We found that the fold expansion was
+identical in all the blocked groups vs the unblocked control group
+(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total
+and across the CD4/CD8 compartments) was not significantly different between any
+of the groups (\cref{fig:inegrin_1_mem}). We also noted that \gls{a2b1} and
+\gls{a2b2} were present on the surface of a significant subset of T cells at day
+6, showing that the target we wished to block was present
+(\cref{fig:inegrin_1_cd49}).
+
 \begin{figure*}[ht!]
   \begingroup
 
@@ -2311,8 +2336,33 @@ do the inverse.
   \label{fig:integrin_2}
 \end{figure*}
 
+Since this last experiment gave a negative result, we decided to hit \gls{a2b1}
+and \gls{a2b2} harder by adding blocking \glspl{mab} at more timepoints between
+day 0 and day 6, hypothesizing that the majority of the signaling would be
+during the period of culture where the \gls{dms} surface concentration was at
+its maximum. Once again, we observed no difference between any of the blocked
+conditions and the unblocked controls in regard to expansion
+(\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total,
+CD4, or CD8) were statistically different between groups
+(\cref{fig:inegrin_2_mem}).
+
+Taken together, these data suggest that the advantage of the \gls{dms} platform
+is not due to signaling through \gls{a2b1} or \gls{a2b2}.
+
 \subsection{blocking IL15 signaling does not alter expansion or phenotype}
 
+% BACKGROUND why is IL15 important?
+
+% TODO cite the luminex data
+\gls{il15} is a cytokine responsible for memory T cell survival and maintenance.
+Furthermore, we observed in other experiments that it is secreted to a much
+greater extend in \gls{dms} compared to bead cultures. One of our driving
+hypotheses in designing the \gls{dms} system was that the higher cell density
+would lead to greater local signaling. Since we observed higher \ptmemp{} across
+many conditions, we hypothesized that \gls{il15} may be responsible for this,
+and further that the unique \textit{cis/trans} activity of \gls{il15} may be
+more active in the \gls{dms} system due to higher cell density.
+
 % TODO actually add captions
 % TODO add fold change and viability to these
 % TODO maybe combine these
@@ -2332,6 +2382,20 @@ do the inverse.
   \label{fig:il15_1}
 \end{figure*}
 
+% TODO how did I determine how much to add?
+% TODO how often was this added?
+% TODO just gate these as normal because this looks sketchy
+We first tested this hypothesis by blocking \gls{il15r} with either a specific
+\gls{mab} or an \gls{igg} isotype control. We observed no difference in the
+expansion rate of blocked or unblocked cells (this experiment also had
+bead-based groups but they did not expand well and thus were not included)
+(\cref{fig:il15_1_fc}). Furthermore, there were no differences in viability
+between any group (\cref{fig:il15_1_viability}). We also performed flow
+cytometry to asses the \ptmemp{} and \pthp{} outputs. Without even gating the
+samples, simply lining up their histograms showed no difference between any of
+the markers, and by extension showing no difference in phenotype
+(\cref{fig:il15_1_mem}).
+
 \begin{figure*}[ht!]
   \begingroup
 
@@ -2347,6 +2411,16 @@ do the inverse.
   \label{fig:il15_2}
 \end{figure*}
 
+We next tried blocking soluble \gls{il15} itself using either a \gls{mab} or an
+\gls{igg} isotype control. Similarly, we observed no difference between fold
+change, viability, or marker histograms between any of these markers, showing
+that blocking \gls{il15} led to no difference in growth or phenotype.
+
+% TODO this can probably be worded more specifically in terms of the cis/trans
+% action of IL15
+In summary, this data did not support the hypothesis that the \gls{dms} platform
+gains its advantages via the \gls{il15} pathway.
+
 \section{discussion}
 
 \chapter{aim 3}\label{aim3}